DESCRIPTION: Understanding dioxygen activation by the cytochrome P450 mono-oxygenases requires knowledge of structure at the molecular level. Serving both beneficial and harmful roles in membrane detoxification and carcinogen activation respectively, the cytochromes P450 have been intensively studied. Pseudomonas putida P450-CAM is the best understood P450 in terms of structure and function. The structures of four P450 reaction states (two ferric, deoxyferrous and oxyferrous) have been established. Following second electron transfer, ferric -peroxide and oxo-ferryl adducts have been proposed as intermediates, the latter as the active oxygen catalyst. To observe and spectroscopically characterize such species for the first time, two approaches will be pursued: the use of "slow substrates" blocked at the normal reaction site to impede oxygen transfer and the use of rapid, photointitiated electron transfer to speed electron delivery. This two prong approach will enhance the likelihood of observing the elusive intermediate. In parallel, X-ray absorption spectroscopy will be used to structurally characterize oxo-ferryl states of chloroperoxidase (CPO) and ferric and ferrous states of nitric oxide synthase (NOS). CPO is the only heme protein clearly established to form thiolate-ligated oxo-ferryl intermediates such as that proposed for P450. Like P450, NOS is a thiolate-ligated heme enzyme. Determination of accurate Fe-S(Cys), Fe-N(porph) and Fe=O bond lengths in the CPO and NOS systems will provide structural information directly applicable to the P450 systems also under study. In addition, will be developed the camphor hydroxylating P450-CAM as a versatile enzyme-based model system for mechanistic studies of important P450 reaction types that are difficult to study due to the insolubility and/or complexity of the natural systems. Dr. Dawson will synthesize appropriately functionalized camphor analogues to use as substrates to customize the model system for each reaction type. The three reactions to be examined are: the C-C bond cleaving deformylation reaction, a reaction of great importance in steroid biosynthesis; the second step in the synthesis of the essential biomolecule nitric oxide (NO) in which NO is produced from arginine by nitric oxide synthase; and the P450-catalyzed conversion of nitosamines to NO dealkylated amines, formaldehyde and carinogenic diazoalkanes. The interplay of structure and mechanism is a common theme in all of the proposed work.